It has been more than 20 years since the bacterial endotoxin test was included in the 1990 edition of Chinese Pharmacopoeia. It is very important to understand the contents of Pharmacopoeia correctly. This article is the author's understanding or idea of bacterial endotoxin test. It is necessary to discuss these related issues for reference.
1. Is it the best choice to test the sensitivity of tachypleus amebocyte lysate in accordance with the bacterial endotoxin limit of the sample? For 10% glucose injection, 0.50EU/ml tachypleus amebocyte lysate was used, and 0.25EU/ml tachypleus amebocyte lysate was used for injection water.
No The sensitivity value of the limulus lysate reagent is equal to the limit of bacterial endotoxin in the sample solution. From the general point of view, the only advantage of the limulus lysate reagent is that the dilution of the sample can be saved. However, there are two disadvantages: 1) because of the influence of solute concentration and PH value, the difficulty of PPC tube operation is increased; 2) it is unavoidable that factors such as retained filter material in sample solution, or unpredictable pipeline dissolution may cause the non-condensation of PPC tube, leading to ineffective test, and the result is "the best prescription". The case, on the contrary, causes waste of time, reagents, drugs and so on, and delays work. Comrades who have done the bacterial endotoxin test know that double dilution method is the simplest and most standardized method to make positive control (PPC) tubes of test samples. Therefore, in order to reduce the interference of sample solution on bacterial endotoxin test, it is suggested to select a more sensitive limulus reagent such as 0.125 EU/ml and dilute the sample solution properly.
2. What is the sampling/testing quantity of bacterial endotoxin test?
Samples for endotoxin test are usually sampled according to the principle of random sampling, unless otherwise specified. In random sampling, special attention should be paid to batches. In the case of multiple sterilization cabinets/batches in a preparation batch, endotoxin test should be carried out for each sterilization cabinet/batch separately.
In the endotoxin test, the same batch of samples but different bottles were tested with the same batch of Limulus reagents from the same manufacturer, and the different results could be repeated even if the replacement and replacement of Limulus reagents were used. After eliminating the possibility of contamination, the bacterial endotoxin contamination of the sample was determined. It was found that the difference of bacterial endotoxin contamination between the two large infusion bottles was more than four times. It can be seen that the difference of bacterial endotoxin contamination between bottles exists objectively. This phenomenon also reminds us that the endotoxin test can not effectively detect the defect of low pollution probability due to the sampling principle. In accordance with the principle of random sampling, we should also consider the production process of the product and the way of endotoxin pollution, and select the typical products at the time node for inspection, so as to avoid the missed detection of pyrogen reaction.
3. Is it necessary to purchase water for bacterial endotoxin test?
Article 22 of the 2010 edition of the Chinese Pharmacopoeia stipulates that "test water refers to purified water unless otherwise specified." At the same time, the 2010 edition of China Pharmacopoeia and China Pharmaceutical Inspection Standard Operating Standards clearly stipulate that "Bacterial Endotoxin Inspection Water" must be used in Bacterial Endotoxin Inspection Experiments, so we can not replace "Bacterial Endotoxin Inspection Water" with sterilized injection water. The bacterial endotoxin test water system refers to Sterile Water for Injection with less endotoxin content (gel method) or 0.005EU/ml (photometric method) and no interference to endotoxin test.
Bacterial endotoxin test water and sterilized injection water have the following differences:
3.1 Endotoxin content: The requirement of sterilized water for injection is lower than 0.25 EU/ml, and the requirement of bacterial endotoxin test water for our company is less than 0.003 EU/ml. Endotoxin test for bacterial endotoxins is negligible in both gel and quantitative methods, but endotoxin can not be ignored in Sterile Water for Injection because of its low endotoxin standard. Sterilized water for injection is used in Limulus test. The superposition of sterilized water for injection and endotoxin in samples may result in false positive results, which will bring unnecessary economic losses to enterprises.
3.2 PH value: The PH value of sterilized water for injection should be 5.0-7.0, and that of bacterial endotoxin test should be 6.0-8.0. Bacterial endotoxin test water PH value 6.0-8.0 is more conducive to the reaction of bacterial endotoxin and Limulus reagent.
3.3 Non-interference: Water requirement for bacterial endotoxin test has no interference effect on endotoxin test, while water for sterile injection has no such standard requirement. Sterilized water for injection may be inhibited, resulting in false negative results. Sometimes endotoxin exceeds the standard and is not detected. Infusion reaction caused by missed detection may bring disaster to enterprises.
4. Does the bacterial endotoxin test for water for injection need to be a positive control for each batch?
To do so, the function of the donor is to verify whether there is a substance inhibiting the coagulation of limulus reagent in the sample, so as to prove that the result of the solution of the sample is accurate. Water for injection is the solvent for the production of injectable drugs. We must ensure the accuracy and reliability of the endotoxin test results of water for injection. If we do not make positive control of the test, once false negative occurs, we can not predict whether the endotoxin in the finished product is qualified. False negative products are still not found in the finished products. The unqualified products inflow into the market will cause serious infusion reaction, which will directly threaten the life safety of clinical patients.
5. After the bacterial endotoxin products are diluted with water for inspection, can the rest of the experiment be preserved and reused?
The diluted endotoxin solution should be restored for more than 10 minutes. Before use, the endotoxin solution should be re-shaken on the scroll mixer for 1 minute. The endotoxin solution should be discarded after more than 4 hours. The diluted endotoxin solution has a lower potency, and its potency usually decreases after long-term storage. Therefore, when it is used again, the experimental results may be biased. Generally, the endotoxin working products are inexpensive and are not recommended for reuse after preservation.
Summary: To ensure the accuracy of bacterial endotoxin test results, three aspects should be done: first, to ensure the reliability of the test equipment, otherwise the experiment will be 100% failure; second, to ensure that the standard dilution is accurate, in order to ensure the accuracy of the experiment results; third, adding samples accurately, which is the basis of the experiment, without this basis, the accuracy of the experiment, Pharmacopoeia provisions, SOP and so on can not be discussed. Up.
Bacterial endotoxin test is related to human life safety. Pharmacopoeia and SOP have strict regulations on its experimental process. Every practitioner should read it carefully, understand it deeply, and understand the reaction principle and influencing factors of tachypleus amebocyte lysate test thoroughly, so as to solve the problems in the experiment, ensure the accuracy of the experimental results and ensure the safety of medication for the wounded and sick.